Four years monitoring of the endangered European plethodontid salamanders

The ongoing biodiversity crisis is strongly threatening amphibians, mostly because of their peculiar physiology, their sensitivity to climate change and the spread of diseases. Effective monitoring involving assessments of pressure effects across time and estimation of population trends play a key role in mitigating amphibian decline. To improve implementation of standardized protocols and conservation efforts, we present here a dataset related to one of the amphibian genera whose onservation status is considered the most declining according to the IUCN. We report information on 66 populations of the endangered European cave salamanders, genus Speleomantes, that was collected through a standardized monitoring along a four-year period (2021–2024). Demographics data of the populations and fitness-related data of single individuals are reported. Furthermore, we include 3,836 high quality images of individuals that can allow to perform studies aiming to assess the phenotypic variability within the genus, and to perform long-term capture-mark-recaptured studies.


Background & Summary
Biodiversity crisis is a major ongoing problem occurring at the global scale, spanning all types of environments [1][2][3] .Such crisis is directly or indirectly enhanced by multiple human activities that determine, or favour, the conditions that pose species at risk of extinction 4 .Among them, environmental changes, and the introduction of new species (either competitors or pathogens) have substantial impacts.In the first case, the relatively fast environmental changes, for example caused by climate change or by pollutants, can quickly create conditions that overcome species physiological limits, preventing their potential gradual adaptation to the novel environmental conditions and driving populations to a dramatic end [5][6][7][8] .In the second case, native species have to deal with newly introduced species that may be stronger competitors 9,10 or that can overcome immunological barriers of individuals 11,12 .All these processes often result in the progressive disappearance of local biodiversity.
Amphibians are the most threatened vertebrates worldwide 11,13 .These vertebrates are generally characterized by a series of specific traits that synergistically enhance their sensitivity to human impacts.For example, many amphibians are characterized by a biphasic life cycle, and their life history stages require both aquatic and terrestrial environments.That means that disturbance in just one of the two environments can provoke significant effects that can lead to the local extinction of the species.Further, amphibians completely rely on the environment for their physiological homeostasis, for example to maintain the body temperature within their optimal range or to balance the water loss [14][15][16] .Considering that most of amphibians are able to exploit a narrow range of environmental conditions 17,18 , even a small change in such conditions can be unbearable.Furthermore, amphibians are often characterized by a low dispersal ability 19 , a characteristic that hampers species to track their preferred conditions, thus exacerbating the impacts of environmental changes.
Long-term monitoring is a necessary prerequisite to set effective protection of amphibians and prevent their declines and extinctions 20 .Repeated surveys are indeed crucial for early identification of population decline and the emergence of potential threats 21 , allowing timely actions and mitigations of related harmful effects 22,23 .With this dataset we provide data from repeated surveys performed on the strictly protected Speleomantes cave salamanders, the only plethodontid species present in Europe 24,25 .There are eight allopatric species of Speleomantes, endemic to specific areas distributed in mainland Italy, in the Republic of San Marino, in south-eastern France and on the island of Sardinia 24,26 .Populations of natural hybrids (between S. ambrosii and S. bianchii) are also known from a small contact zone located in Apuan Alps (Northern Tuscany) 27 .Their overall conservation status is alarming: one species is Near Threatened, one Vulnerable, four Endangered and two Critically Endangered (www.iucnredlist.org;last access on 05 February 2024).A mandatory monitoring activity of protected species has been imposed by the European Union (Article 17 of the Habitats Directive; https://nature-art17.eionet.europa.eu/article17/)with the aim of constantly updating their conservation status; however, very limited data is currently available for the genus Speleomantes.In addition to providing a large amount of data covering a four-year period, this dataset offers the possibility of being combined with those previously published [28][29][30] , allowing the monitoring period for some populations to be extended to seven years.The availability of standardized data from multiple conspecific populations can also increase the possibility of detecting intraspecific variation as key traits.This can contribute to the conservation of not just species, but also of populations and intraspecific diversity [31][32][33]

Methods
The monitoring activity involved multiple surveys on 66 populations of Speleomantes salamanders in the period 2021-2024 (Tables 1 and 2).The surveyed populations were subterranean or fully epigean.In the first case, the subterranean environments (both natural and artificial) were surveyed entirely or up to the point where the exploration required the use of speleological equipment; in surface environments, specific plots delimited the study area.The surveys were carried out producing an average sampling effort of approximately 27 m 2 /minute 34 .Speleomantes were opportunistically searched and captured within the sampling area: these were the active individuals and those spotted hiding under stones or inside crevices.All captured individuals from a population were temporarily placed in disinfected (using bleach) fauna boxes until processing.A portable photo studio has been set in the field which allows high quality images to be obtained from which it is possible to extrapolate biometric data 35,36 .This portable studio is designed to obtain high-quality images by controlling the position of the camera and lights (i.e.flash), allowing individuals to be shot perpendicularly from above and producing comparable images useful for individual recognition through the use of the dorsal pattern 37 .This also allows a high standardization of the coloration of salamanders 35 .During each survey, we shot a first image taken on a Pantone Calibrite ColorChecker Passport Photo 2 (for brevity, hereafter Pantone); this allowed us to obtain the white reference for image calibration, and the length reference to insert within each image a as a unit of measurement.Salamanders were weighed on a digital scale (0.01 g) and visually inspected to record multiple data.
An initial inspection was carried out to assess the presence of ectoparasites, i.e. leeches of the genus Batracobdella, which are known to parasitise the Sardinian Speleomantes 24,38 (Fig. 1).When leeches were present, they were counted and weighed separately.Subsequently, a check was carried out for any malformation (e.g., forked tail, foot shape 39,40 ;) (Fig. 2).In some of the monitored populations, individuals were previously marked using either the Visual Implant Elastomers 41 and Visual Implant Alpha tags 42 , we used an UV light to assess the presence of these tags.Tags were placed on arms, flanks and the base of the tail (for further information on tag implantation refer to 41,42 ).The individuals' head was then checked to assess the presence of the "mental gland", a male sexual character located under the lower jaw 24 ; all salamanders found with this character were considered adult males.Considering the exclusivity of males in showing distinctive morphological characters, adult females and juveniles are generally distinguished based on body size 24 .Speleomantes can lose and regrow their tail to avoid predation 43 ; this makes total length (TL) unreliable for making this distinction.We then used the snout-vent length (SVL, in mm) to size individuals and distinguish between juveniles and adult females 44 .The threshold was set based on the average minimum size observed for gravid females and mature males 45,46 : for the three continental species (S. strinatii, S. ambrosii and S. italicus), hybrids and for S. genei it was set at 50 mm, while for the other "larger" Sardinian species (S. flavus, S. supramontis, S. imperialis and S. sarrabusensis) to 55 mm.This distinction was only possible in the laboratory after estimating the SVL of individuals from the images (see below).
Camera RAW files (.CR2) were opened and processed with Adobe Photoshop.For each population we first opened the Pantone photo with Adobe Camera Raw and set white calibration using the White Balance Tool function; the resulting profile was used to white balance all photos taken during a single session.We then set the scale to 10 mm using the Pantone reference measurement; the same amount of pixels was used for the entire population thanks to the fixed position of the camera 35 .The images were cropped and cleaned by adding a white background.After adding the reference scale they were transformed into JPEG, allowing to reduce the weight of the file (~2-3 MB) without compromising its quality 28 (Fig. 1).We then used Fiji 47 to estimate individuals SVL 36 .After setting the image scale (straight line) via the scale bar, we used the segmented line to draw a line from the snout to the tip of the tail, following the centre of the body; this allowed to measure the individual's total length.Another segmented line was drawn from the tip of the snout to the extremity of the body, that is the point that corresponds to the opening of the cloaca 36 ; this allowed us to estimate SVL.The differences between TL and SVL were used to obtain tail length.

Data Records
The dataset presented here (Speleomantes photographic dataset 2021-2024, available on figshare 48 ) consists of 3,836 high-quality images of individuals from 66 Speleomantes populations, including the eight known species distributed in mainland Italy (including the Republic of San Marino) and Sardinia 24 , as well as hybrid populations occurring in the north (i.e., natural populations) and south (i.e., introduced populations) of Tuscany 27,49 .Detailed information on Speleomantes populations (coded following 28 ) is shown in Tables 1 and 2. Table 1.Summary of the surveys carried out in the period 2021-2024.For each mainland species (including hybrids), the number of populations surveyed, the periods in which surveys were conducted and the total number of individuals photographed are reported.

Species
Each image comes with additional information about the individual's biometric data (e.g., size, weight) and condition (e.g., presence of parasites, malformations) (Information on Speleomantes individuals 48 ).The .CSV supporting dataset is composed as shown in Table 3.

technical Validation
Surveys were performed during a single day and by operators adopting the same sampling effort.The standardisation and quality of images provided here is guaranteed by the adoption of the best protocol described for these species 28,29 .The snout-vent and total length were estimated through analysis of the high-quality images provided here; this methodology allows reliable estimates with very small measurement error (~2 mm) even if measures are taken by a non-expert operator 36 .Potential errors due to measurement or transcription were double checked by plotting the data (SVL vs TL and TL vs weight) (Fig. 3).Anomalous data were compared to paper field data, and dubious measurements were repeated.Considering the SVL threshold used to distinguish juveniles (50 and 55 mm for normal and giant species, respectively), all individuals with SVL lower than the respective threshold were considered juveniles.

Usage Notes
This dataset has broad application.In addition to being an important tool for estimating and monitoring the abundance of populations over time, and for studying demographic parameters (e.g., age distribution, survival, growth rate) as specified above, we list some other important uses here.The conditions of individuals can be evaluated from different points of view: in addition to verifying the presence of ectoparasites and malformations, using the ratio between total length (TL) and weight we can evaluate the body condition of each individual 50 .This fitness-related trait allows inter-and intrapopulation comparisons in space and time 51,52 , making it possible to evaluate factors that negatively influence individual condition 53 .In this circumstance, the use of TL in

Tag
Unique code identifying marked individuals with Alpha Tag or Visual Implant Elastomers.For the latter, the code is composed by: a letter indicating the colour (b = blue, g = green, p = pink, y = yellow) and a series of number indicating the specific body part (see 41    estimating individual body condition is more appropriate, since these salamanders usually accumulate fat in the tail 54 .The images can be used for geometric morphometry 55 , for coloration analyses 47,56,57 , and can also be used to test the reliability of different software for the automatic photographic identification of individuals [58][59][60] .

Fig. 1
Fig. 1 Example of the high-quality images composing the dataset.In this picture an individual of Speleomantes flavus is parasitized by a leech of the genus Batracobdella.Scale bar = 10 mm.

Fig. 2
Fig. 2 Some examples of malformations observed in Speleomantes during our study.
for further information) Sex M = adult male; F = adult female; J = juvenile Eggs Indicates whether developed eggs were visible from salamanders' abdomen Tail issue Indicates whether the tail shows normal (0) or shortened (1) length Leech number and weight Indicates how many leeches were parasitizing the salamander and the overall weight of the parasites Malformation Indicates the presence of a body malformation.This could be related to limb, head, tail.NA = no malformations observed Weight Individual weight (g) SVL Snout-vent length (mm) Tail Tail length (mm) TL Total length (mm) Picture number Identifies the image of the individuals photographed during each survey Scale bar Indicates the length (mm) of the scale bar in images N_operators Indicates the number of researchers involved in salamanders searching activity

Fig. 3
Fig. 3 Linear regression plots used to evaluate the presence of erroneous data.(A) Correlation between SVL and tail length; (B) correlation between TL and weight.

Table 2 .
Summary of the surveys carried out in the period 2021-2024.For each Sardinian species, the number of populations surveyed, the periods in which surveys were conducted and the total number of individuals photographed are reported.

Table 3 .
Description of the accompanying dataset "Information on Speleomantes individuals".This Excel table contains qualitative information of each photo composing the main dataset.